Aminoglycoside antibiotic kanamycin functionalized tetraphenylethylene molecular probe for highly selective detection of bovine serum albumin protein

A novel tetraphenylethylene (TPE) functionalized aminoglycoside antibiotic kanamycin (TPE-kana 1) has been successfully synthesized and characterized by means of modern analytical and spectroscopic techniques. The probe TPE-kana 1 showed strong affinity towards bovine serum albumin (BSA) compared to its other biological competitors. The recognition of BSA have been investigated employing UV–Vis absorption and fluorescence emission spectroscopy. The significant color change of TPE-kana 1 with BSA can be observed by necked eye, where the role of AIE-active TPE molecule is handle in both optical and colorimetric changes. The quenching of fluorescence of TPE-kana 1 with BSA was characterized by fluorescence spectroscopy, with 71.16% of quenching efficiency. Moreover, the Stern–Volmer quenching constant was calculated and found to be 2.46 × 107 M−1. Probe TPE-kana 1 showed detection limit of 2.87 nM (nM) towards BSA with binding constant 7.56 × 107 M. A molecular docking study is also performed to investigate the detail interactions between TPE-kana 1 with the sites of BSA via non-covalent i.e., H-bonding, π-cation interactions, π-donor hydrogen bonds and π-π interactions. The lowest binding energy conformation was found at − 10.42 kcal/mol.

www.nature.com/scientificreports/ II [17][18][19][20][21] . Therefore, there is a need to develop a new, highly efficient chemosensors for the selective detection of proteins are on great demand. On other hand, aminoglycoside antibiotics have possessed unique structurally features with number of amino and hydroxyl group. The presence of multiple amino groups on kanamycin allows it to modify through amidation reactions. The amino group at 6′ position of kanamycin is more reactive and undergoes site selective amidation, compared to other amino group due to steric hindrance factor 22 . Various amide conjugated biomaterials have been reported for different biological applications such material design, sensing of biomolecules and biomedical applications [23][24][25][26] . The molecular modelling and functionalization of aminoglycosides through covalent and non-covalent interactions have been used for the fabrications of various types of biomaterials such as hydrogels, nanoparticles, chemosensor, biointerfaces, amphiphiles and microstructures 15,[27][28][29][30] . In our recent work, we have also shown that the self-assembled flower-like microstructure due to coordination of primary amino and hydroxyl group of kanamycin with Cu 2+ ions and furthermore we have shown that flower shown to be having high surface areas, thus, used as a photocatalyst for the degradation of organic dyes 31 .
In this work, we described the synthesis (over four steps, Fig. 1) and photochemical properties of first ever TPE-functionalized kanamycin (kana) antibiotic (coded as: TPE-kana 1), a strong fluorescent chromophore for selective detection of BSA via supramolecular interactions. The detection of BSA using AIE-active TPE-kana 1 in aqueous solution was found to be most powerful tool due to its easy preparation, rapid response, excellent sensitivity, high selectivity, and non-destructive nature, as the responses of TPE-kana 1 towards various other biological competitors are insignificant compared to the BSA.

Results and discussion
Design and synthesis of TPE-kana 1. In the present study, kanamycin, an aminoglycoside antibiotic functionalized with highly fluorescent 1,1,2,2-tetraphenylethene (TPE) molecule (emits white light), was successfully synthesized. Fluorescent TPE-functionalized kanamycin TPE-kana 1 was then used for the selective detection of BSA. The TPE-kana 1 conjugate was synthesized by a multistep reaction strategy as illustrated in Fig. 1. The (2-(4-nitrophenyl-1,1,2-triyl)tribenzene 3 was synthesized in the first step by mono-nitration of TPE 1. The 4-(1,2,2-triphenylvinyl)aniline 4 was obtained by reducing compound 3 using hydrazine hydrate. The compound 4 was then heated with succinic anhydride in toluene yielded 4-oxo-4-((4-(1,2,2-triphenyl)phenyl) amino)butanoic acid 5, the compound 5 was then upon amide coupling with kanamycin antibiotic resulted in the formation of target compound TPE-kana 1 (70% yield). All the compounds were confirmed by 1 H NMR spectroscopy and the final target molecule was characterized by IR, NMR, HRMS sophisticated techniques and purity was confirmed by high-pressure liquid chromatography (HPLC), and also optical rotation as illustrated in  Optical properties of TPE-kana 1 at different pH. The pH (4.0, 7.0 and 9.2) dependent changes of TPE-kana 1 in water were investigated using UV-Vis absorption and fluorescence spectroscopy (Fig. S10). In UV-Vis absorption spectra TPE-kana 1 showed absorption band at 310 nm, whereas there are not much differences in the absorption spectra of TPE-kana 1 at different pH. Moreover, in fluorescence emission spectra of TPE-kana 1 almost negligible emission observed at ph-4, moderate emission at pH-9.2 and the highest emission intensity was observed at pH-7. The TPE-kana 1 possess many hydroxyls (-OH) and primary amino (-NH 2 ) group of kanamycin functionalized TPE as a AIE active moiety. The presence of multiple -OH and amine -NH 2 group of kanamycin and TPE group leads to such different absorption and emission behavior of TPE-kana 1. The multiple -OH and amine -NH 2 group kanamycin could provide utmost opportunities for the formation of both non-covalent interactions at different pH which may leads to construction of versatile biomaterial framework. Moreover, this different behavior of TPE-kana 1 at different pH was also characterized using scanning electron microscopy (SEM) which shows almost similar mode of aggregations at pH-4 and 9.2, a globular particular aggregates like structure of TPE-kana 1 was observed. Whereas at pH-7 the TPE-kana 1 produces leaf-like structures ( Fig. S11) and in pure THF solvent TPE-kana 1 self-assembled into globular structure (Fig. S12).

Sensing performance of TPE-kana 1
The stock solution of the TPE-kana 1 (5 × 10 −5 mol/L) was prepared by dissolving in DMSO solvent. The stock solution from DMSO was used to prepare solution of TPE-kana 1 in distilled water. A series of biomolecules (4 equiv.) such as BSA, heparin, ascorbic acid, sodium pyrophosphate, sodium oxalate, sodium citrate, adenosine triphosphate, glutamic acid, aspartic acid, sodium acetate, and glucose were added to TPE-kana 1 in water. The sensing performance of TPE-kana 1 was then monitored by the color change in the UV light (365 nm) and is depicted in Fig. 3. Under UV light the blue fluorescence of the blank changes to slightly green in the presence of BSA, whereas TPE-kana 1 did not show such color change in presence of other biomolecules, as shown in Fig. 3. These naked eye results suggested that TPE-kana 1 as a fluorescent sensor can be used for selective detection of BSA in solution. Furthermore, we have examined sensing capability of TPE-kana 1 in distilled water using UV-Vis absorption, fluorescence spectroscopy and also studied competitive experiments to figure out the selectivity of TPE-kana 1 toward BSA. The theoretical docking studies employed to investigate interaction of TPE-kana 1 with BSA either site I and/or site II or within them.

UV-Vis absorption study.
The UV-Vis absorption spectroscopy was employed to investigate the sensing response of TPE-kana 1 towards biomolecules. TPE-kana 1 shows selectivity towards BSA protein only as compared with other biomolecules/proteins as shown in Fig. 4a. Typically, in aqueous solution TPE-kana 1 (5 × 10 −5 M) exhibit absorption maxima centred at 306 nm, and which upon BSA addition a new absorption band appeared near 280 nm wavelength with the enhanced intensity and broaden peak at 306 nm and no such effect occurred with various other biological competitors as shown in Fig. 4a. These results clearly demonstrated that the non-covalent interactions between TPE-kana 1 with BSA not only through intermolecular hydrogenbonding between the amino-and hydroxyl-functional group of TPE-kana 1 with BSA but also packing of the TPE-kana 1 in the pocket of BSA, which leads to change in the optical properties of TPE-kana 1. For a detailed understanding, titration of TPE-kana 1 with BSA (0-120 nm) in distilled water (D/W) was evaluated, which was   The emission intensity at 476 nm considerably decreased from 422 to 107 as the concentration of BSA increased from 0 to 120 nM and further additional of BSA does not show any more changes may be due to the detection limit. This clearly suggest the interaction of TPE-kana 1 with BSA protein over other biological competitors almost silent, because of hydrophobic interactions along with H-bonding between -OH and -NH 2 group of kana moiety and amino acids of BSA followed by π-π interactions between tryptophan amino acid of BSA and TPE unit of TPE-kana 1. We believe that the strong H-bonding between kanamycin of TPE-kana 1 bind within the molecular cleft of BSA, which can be clearly seen in changes under UV-Vis absorption (Fig. 4) and fluorescence emission spectroscopy (Fig. 5) and later docking study confirm interactions. The initial fluorescence intensity of TPE-kana 1 was found to show a significant decrease upon incremental addition of the BSA. Therefore, fluorescence quenching efficiency (η) was calculated using equation [(I 0 − I)/ I 0 ] × 100%, where I 0 and I are the fluorescence intensities before and after addition of the BSA. After the addition  . Emission spectra of probe 1 (5 × 10 −5 M) in water with the addition of (a) 120 nM of BSA, heparin, ascorbic acid, sodium pyro-phosphate, sodium oxalate, sodium citrate, adenosine triphosphate, glutamic acid, aspartic acid, sodium acetate and glucose (b) addition of BSA (0-120 nM). Selectivity study of 1 for BSA. To evaluate the selectivity study of TPE-kana 1 for BSA detection, the fluorescence response of TPE-kana 1 with several anions (120 nM) as well as biological competitors (120 nM) of BSA such as heparin, ascorbic acid, sodium pyrophosphate, sodium oxalate, sodium citrate, adenosine triphosphate, glutamic acid, aspartic acid, sodium acetate, glucose was investigated. TPE-kana 1 did not show a considerable response neither to the anions and not to biological competitors to BSA as illustrated in Fig. 6. It clearly shows selective detection of BSA over other biological competitors.  Fig. 7a, it can be seen that Stern-Volmer plot followed a good linearity at low concentrations of BSA whereas, at higher concentrations, the linearity slightly deviated as an upwardly bent curve. The K sv value obtained for BSA was 2.46 × 10 7 M −1 . This indicated that the BSA exhibited exclusive quenching ability towards fluorescent TPE-kana 1 in water.   Figure 6. Fluorescence emission responses of TPE-kana 1 toward BSA (120 nM) and its other biological competitors (120 nM) at 476 nm in D/W. www.nature.com/scientificreports/ To evaluate the detection limit, emission titration of TPE-kana 1 with BSA in distilled water was carried out by adding increasing concentrations of BSA solution (0-120 nM) and the emission intensity as a function of BSA added was then plotted, Fig. 7b. The detection limit was calculated by using the equation as follows:

Stern-
Detection limit = 3 σ/m. Where σ is the standard deviation of the emission of the free sensor and m is the slope between emission at 476 nm and concentration of BSA. The detection limit for BSA was shown to be 2.87 nM (R 2 = 0.907) in distilled water showing the fluorescence intensity of TPE-kana 1 at 476 nm (λ ex = 310 nm) as a function of BSA concentration. Hence the detection limit of TPE-kana 1 in distilled water was found to be 2.87 nM which is higher or comparable with other molecules used for detection of BSA (Table S1).
Binding constant. Furthermore, the Benesi-Hildebrand plot (ESI Fig. S14), was used to define the binding constant (K a ) between 1 and BSA. The linear relationship of absorption intensity as a function of 1/[BSA] was found to be 7.56 × 10 7 M with R 2 = 0.9968. This suggest that the TPE-kana 1 to have strong binding affinity towards BSA. Furthermore, SEM images clearly shows strong interaction between TPE-kana 1 with BSA, which produces highly packed coagulated microstructure (Fig. S15) as compared with TPE-kana 1 only produces particular aggregates (Fig. S13) in in distilled water.

Molecular docking study
To evaluate binding mode of TPE-kana 1 with BSA, molecular docking study using AutoDock4.2.6 32 was employed. The TPE-kana 1 shows significant binding affinity with the BSA protein and the lowest binding energy conformation was found at − 10.42 kcal/mol and shown in Fig. 8. The binding site of TPE-kana 1 at the sub-domains IIA of site 1 was analyzed with the least binding energy conformation of TPE-kana 1. To understand the bonding and non-bonding interactions of TPE-kana 1 with BSA, we further analyzed the docked complex. The analysis of BSA with TPE-kana 1 complex shows the conventional hydrogen bonding interactions with BSA residues such as Glu443 (2.01 Å, 1.84 Å, 1.73 Å, 2.96 Å), Lys294 (2.21 Å), Arg217 (1.67 Å) as shown in Fig. 8a. Lys439, Glu293 forms non-conventional hydrogen bonding interaction with TPE-kana 1 as shown in Fig. 8a. In addition, Arg198, and Arg256 forms π-Cation, Tyr149 forms π-π type of interaction, Leu237, and Ala290 forms π-type of interactions, Trp-213 forms π-donor hydrogen bond as shown in Fig. 8a. Whereas, Glu152, Ser191, Arg194, Leu218, Phe222, Val240, His241, Leu259, Ser286, Ile289, Glu291, and Tyr451as shown in Fig. 8b. As shown in Fig. 9, TPE-kana 1 forms conventional hydrogen bonding, carbon-hydrogen, van der Waals, and π-type of interactions play the major role for the binding pocket residue of BSA protein.
In summary, we have successfully designed and synthesized TPE-functionalized kanamycin antibiotic TPEkana 1 as a molecular probe for selective and sensitive detection of BSA. TPE-kana 1 showed significant AIE properties in THF:water solvent systems. Furthermore, TPE-kana 1 exhibited BSA induced decreased fluorescence emission intensity in distilled water compared to other examined anions and biological competitors in aqueous medium. The sensing of BSA was well characterized by using UV-Vis absorption and fluorescence emission spectroscopy, the quenching of fluorescence observed with BSA. Moreover, the quenching efficiency was calculated and obtained was 71.16%. The Stern Volmer quenching constant was calculated and found to

Experimental section
Materials and methods. Compounds 3 and 4 were synthesized using reported literature procedure and compound 5 and TPE-kana 1 were synthesized using following procedure (Fig. S1). All reagents including toluene, DMSO, acetic acid, hydrazine hydrate, O-(Benzotriazol-1-yl)-N,N,N′,N′-bis(tetramethylene)uronium hexafluorophosphate (HBPyU), Tetrahydrofuran (THF), and kanamycin sulfate and various biomolecules such as BSA, heparin, ascorbic acid, sodium pyrophosphate, sodium oxalate, sodium citrate, adenosine triphosphate, Glutamic acid, aspartic acid, sodium acetate, and glucose were purchased from Sigma-Aldrich, and TCI chemicals. All the reactions were monitored by thin-layer chromatography (TLC) and 1 H and 13 C NMR spectra were recorded on 400 MHz and 100 MHz Bruker spectrometer with tetramethylsilane (TMS) as an internal standard and CDCl3-d and MeOD-d4 as deuterated solvents. Thermofisher exactive orbitrap MALDI-TOF measurements were used for HRMS, a Schimadzu Biotech Axima performance spectroscopic instrument. UV-Vis absorption spectra were recorded using a UV-Vis 1800 Shimadzu spectrophotometer and fluorescence emission was measured on an Agilent, Carry Eclipse spectrofluorophotometer.
General procedure for UV-Vis and fluorescence measurements in aqueous solutions. A stock solution (1 mM) of TPE-kana 1 was prepared in DMSO and was stored in a cold and dark place. The stock solution was used for the experiments after appropriate dilution in distilled water (D/W). The concentration of TPE-kana 1 was confirmed by absorbance at 306 nm for the TPE fluorophore UV-Vis absorption spectra (250-550 nm) of the samples in a 10 mm path length cuvette was measured using a UV-Vis 1800 Shimadzu spectrophotometer fluorescence emission spectra of TPE-kana 1 in a 10 mm path length cuvette were measured using an Agilent, Carry Eclipse Spectro fluorophotometer with excitation at 310 nm. All absorbance and fluorescence measurements were carried out in 100% aqueous solutions at room temperature.  www.nature.com/scientificreports/ spectra were recorded with the addition of BSA and its other competitor's different anions (20 × 10 −3 M). The absorption and emission spectra were completely recorded at room temperature.

UV-vis absorption and fluorescence emission titration of the TPE-kana 1 upon the addition of BSA:
The 2 mL TPE-kana 1 solution (5 × 10 −5 mol/L) in D/W was placed in the quartz cell and the fraction of BSA solution (0-120 nM) was added, and the corresponding absorption and emission spectra were recorded at room temperature.
Naked-eye detection. The DMSO stock solution of the TPE-kana 1 (5 × 10 −5 mol/L) was used to prepare solution for necked eye experiments by dissolving it in D/W. Then BSA and its other competitors such as heparin, ascorbic acid, sodium pyrophosphate, sodium oxalate, sodium citrate, adenosine triphosphate, Glutamic acid, aspartic acid, sodium acetate, and glucose were added, the photograph were taken under 365 nm UV light.
Limit of detection. To determine the detection limit, emission titration of TPE-kana 1 with BSA was carried out by adding increasing concentrations of BSA solution (0-120 nM) and the emission intensity as a function of BSA added was then plotted.

Molecular docking study.
To investigate the binding mode of TPE-kana 1 with BSA, we employed molecular docking calculations using AutoDock4.2.6 software (https:// autod ock. scrip ps. edu/) 35. The crystal structure of BSA was retrieved from the protein database (source code: 4OR0.pdb). Here, chain A was considered for the molecular docking study. The three-dimensional atomic co-ordinate of TPE-kana 1 was generated using the Discovery Studio Visualizer 35 . For the docking study, a grid box size of 80 × 80 × 80 with a spacing of 0.375 Å was built around the active site of BSA which is present in between the domain IIA and IIIA. Next, we employed a local docking protocol, to explore a binding mode of TPE-kana 1, similar to an earlier study 34 . Here, we keep BSA as rigid and TPE-kana 1 as a flexible molecule. The output docking conformations were generated by applying the Lamarckian Genetic Algorithm (LGA). These output conformations were further clustered using an all-atom RMSD with a cut-off of 4 Å. The clusters were further analyzed based upon binding, van der Waals, and electrostatic energy, etc. The lowest binding energy conformation of TPE-kana 1 was further analyzed for the bonding interactions using PyMol (DeLano 2002) and Discovery Studio visualizer 35 , respectively.